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More than 50% of patients with chondrosarcomas exhibit gain-of-function mutations in either isocitrate
dehydrogenase 1 (IDH1) or IDH2. In this study, we performed genome-wide CpG methylation sequencing of
chondrosarcoma biopsies and found that IDH mutations were associated with DNA hypermethylation at CpG
islands but not other genomic regions. Regions of CpG island hypermethylation were enriched for genes
implicated in stem cell maintenance/differentiation and lineage specification. In murine 10T1/2 mesenchymal
progenitor cells, expression of mutant IDH2 led to DNA hypermethylation and an impairment in differentiation
that could be reversed by treatment with DNA-hypomethylating agents. Introduction of mutant IDH2 also
induced loss of contact inhibition and generated undifferentiated sarcomas in vivo. The oncogenic potential of
mutant IDH2 correlated with the ability to produce 2-hydroxyglutarate. Together, these data demonstrate that
neomorphic IDH2 mutations can be oncogenic in mesenchymal cells.
[Keywords: isocitrate dehydrogenase mutation; 2-hydroxyglutarate; differentiation; contact inhibition; tumorigenesis;
DNA methylation; chondrosarcoma]
Supplemental material is available for this article.
Received July 16, 2013; revised version accepted August 16, 2013.
11
12
1986
Cytosolic IDH1 and mitochondrial IDH2 are NADP+dependent enzymes that metabolize isocitrate to a-ketoglutarate (aKG). Frequent somatic mutations of IDH1 and
IDH2 were initially identified in ;80% of intermediategrade gliomas (Yan et al. 2009) and ;20% of de novo acute
myeloid leukemias (AMLs) (Mardis et al. 2009; Ward et al.
2010). More recently, they were also found in more than
half of patients with chondrosarcomas (Amary et al.
2011a) and skeletal disorders characterized by cartilage
tumors (Amary et al. 2011b; Pansuriya et al. 2011). Almost
all mutations observed in IDH1 and IDH2 are monoallelic
point mutations affecting only a few residues, suggesting
that they are unlikely to be loss of function. Indeed,
metabolomic and biochemical analysis revealed that mutant IDH enzymes gain a neomorphic activity of producing
2-hydroxyglutarate (2HG) from aKG (Dang et al. 2009;
Ward et al. 2010). 2HG is normally present at very low
levels in cells but exhibits a >100-fold increase in tumor
samples with IDH mutations. It is believed that IDH
mutations promote tumorigenesis through accumulating
the putative oncometabolite 2HG.
GENES & DEVELOPMENT 27:19861998 2013, Published by Cold Spring Harbor Laboratory Press; ISSN 0890-9369/13; www.genesdev.org
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Results
Genome-wide DNA methylation landscape of IDH
mutant chondrosarcomas
A panel of snap-frozen surgical specimens from 21 patients with chondrosarcomas was collected through an
institutional review board-approved protocol. Targeted
sequencing results revealed an ;50% frequency of IDH
mutations in chondrosarcomas (seven of the samples
had the R132 IDH1 mutation, three had the R172 IDH2
mutation, and 11 were wild type for IDH1/2), consistent
with previous reports (Amary et al. 2011a; Pansuriya et al.
2011; Arai et al. 2012). Compared with samples that were
wild type for IDH1/2, IDH1 or IDH2 mutant samples
1987
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Lu et al.
Genomic DNA of 10T cells was extracted and processed for ERRBS to generate highly reproducible basepair resolution DNA methylation profiles (Supplemental
Fig. S3A). In agreement with results from chondrosarcoma biopsies, IDH2 R172K mutant cells compared
with wild-type cells showed a profound DNA hypermethylation at CpG islands across all chromosomes
(Fig. 2A). In contrast, significantly less differentially
methylated CpGs were observed between wild-type
IDH2 and vector cells, with even distribution of hypermethylated and hypomethylated sites (Fig. 2A). Increased methylation at several histone marks, such as
H3K9me3, H3K9me2, and H3K4me3, was also found in
IDH2 R172K mutant cells, which could reinforce with
DNA methylation to modulate gene expression (Supplemental Fig. S3B).
In total, there were 2400 genes with differentially
methylated CpGs at their promoters, and a predominance
in DNA hypermethylation was observed when comparing the IDH2 R172K mutant with wild-type cells (78.1%
being hypermethylated, P-value < 0.0001) (Supplemental
Table S3). To gain more insights into the mechanism of
aberrant DNA methylation, the group of genes that were
promoter DNA-hypermethylated in IDH2 R172K mutant cells was subjected to gene set enrichment analysis
(GSEA) with the Broad Institute Molecular Signatures
Database (http://www.broadinstitute.org/gsea/msigdb/
index.jsp), which includes >3000 curated gene sets collected from various research sources. The top four most
statistically significantly enriched gene sets were Polycomb-repressive complex 2 (PRC2) target genes or genes
marked by H3K27me3 in embryonic stem cells (Fig. 2B;
Supplemental Table S4). Notably, recent genome-wide
mapping of Tet1-binding sites revealed that >95% of
PRC2 target genes were bound by Tet1 (Wu and Zhang
2011), and Tet1 has been proposed as a guardian to protect
these regions from accidental DNA methylation (Williams
et al. 2012). Taken together, the DNA methylome analysis
suggests that acquisition of an IDH2 R172 mutation is
sufficient to establish DNA hypermethylation, the pattern
of which matches the patterns of Tet1- and PRC2-binding
activity.
1988
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Figure 2. Mutant IDH2 induces CpG island hypermethylation phenotype. (A) Stacking bar graph showing percentage of hypermethylated and hypomethylated CpGs of all CpGs located in CpG islands for each chromosome, comparing IDH2 R172K mutant with
wild-type (WT) cells (left) or vector with IDH2 wild-type cells (right). Green represents proportion of hypomethylated cytosines, and
magenta represents hypermethylated ones. Only CpGs with a Q-value <0.01 and a methylation difference of at least 25% are shown. (B)
GSEA was performed on genes that were promoter DNA-hypermethylated in IDH2 R172K mutant cells. The table shows the top four
most significantly enriched gene sets from the Broad Institute database and their P-values.
1989
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Lu et al.
Figure 3. IDH2 mutation inhibits mesenchymal differentiation. (A) Vector (Vec), wild-type (WT), or R172K mutant IDH2 cells were
treated with adipocyte differentiation cocktail. After 7 d of differentiation induction, representative microscopic images of cell
morphology were recorded, and mRNA expression of Adipoq and Fabp4 was measured by quantitative real-time PCR (qRT-PCR). (B)
Vector, wild-type, or R172K mutant IDH2 cells were treated with chondrocyte differentiation cocktail. After 10 d of differentiation
induction, representative microscopic images of cell morphology were recorded (arrowheads point to mature chondrocyte-resembling
cells), and mRNA expression of Acan and Col2a1 was measured by qRT-PCR. (C) 10T vector, wild-type, or R172K mutant IDH2 cells
were treated with adipocyte or chondrocyte differentiation cocktails. Cell numbers were counted at days 0, 3, and 6 after differentiation
induction. (D) Six days after adipocyte or chondrocyte differentiation induction, 10T vector, wild-type, or R172K mutant IDH2 cells
were lysed, and protein levels of cyclin D1 were measured by Western blot. Tubulin was used as loading control. For all experiments,
the average 6 SD from three biological replicates are shown.
1990
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Figure 4. Differentiation impairment by mutant IDH2 correlates with 2HG production. (A) Structural modeling of IDH2 catalytic site
showing Arg 172 and Ala 174. Isocitrate carbons are in yellow except carbon 6 containing the b-carboxyl, which is highlighted in cyan.
Carbon atoms of amino acids (green), amines (blue), and oxygens (red) are also depicted. Hydrogen atoms are omitted for clarity. Dashed
interactions corresponding to hydrogen and ionic bonds. The prime (9) denotes that the residue comes from the other
lines show <3.1 A
monomer of the IDH dimer. (B) 10T cells expressing vector (Vec), wild-type (WT), R172K, R172K/A174D, or R140Q mutant IDH2 were
lysed, and IDH2 expression was measured by Western blot. 2HG levels were measured by GC-MS and normalized to internal standard
(D5-2HG) and cell number. (C) 10T cells expressing wild-type or various mutant IDH2 were treated with adipocyte or chondrocyte
differentiation cocktails. mRNA expression of Adipoq, Fabp4, Acan, and Col2a1 was measured by qRT-PCR after 8 d of differentiation
induction. For all experiments, the average 6 SD from three biological replicates are shown.
To functionally link DNA hypermethylation to differentiation impairment, IDH2 R172K mutant cells were
treated with 5-azacytidine (5-aza) to test whether inhibiting DNA methyltransferase had any effect on reversing
the differentiation inhibition by mutant IDH2. After
treatment with low doses of 5-aza for 48 h to induce
global DNA demethylation (Supplemental Fig. S4), IDH2
R172K mutant cells were allowed to proliferate in the
absence of 5-aza until reaching confluence before being
subjected to adipocyte differentiation induction. Compared with untreated cells, a transient exposure to 5-aza
led to the accumulation of lipid droplets and a dosedependent increase in the expression of mature adipocyte
1991
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Lu et al.
Figure 5. DNA-hypomethylating agent reverses the differentiation defect in mutant IDH2 cells. (A) After 8 d of adipocyte
differentiation induction, microscopic images of cell morphology were recorded in IDH2 R172K mutant cells with or without
transient 5-aza treatment. (B) After 8 d of adipocyte differentiation induction, mRNA expression of Adipoq and Fabp4 in
IDH2 R172K mutant cells with or without transient 5-aza
treatment was measured by qRT-PCR. The average 6 SD from
three biological replicates are shown.
1992
Given the malignant phenotypes of differentiation blockade and loss of contact inhibition observed in vitro,
xenograft studies were performed to test whether 10T
cells with IDH2 mutation could generate tumors in vivo.
IDH2 R172K mutant cells formed palpable tumors 20
d after subcutaneous injection and continued to grow up
to 1000 mm3, while vector and wild-type IDH2 cells
showed no tumorigenicity over the course of study (Fig.
7A). Immunohistochemistry staining revealed that these
tumors resembled poorly differentiated sarcomas with a
high Ki67 index (Fig. 7B). Consistent with in vitro studies,
these tumors also had high levels of cyclin D1, low p27
expression (Fig. 7B), and no detectable staining for mature
mesenchymal lineage markers (data not shown). Tumors
from IDH2 R172K mutant cells showed 2HG levels that
were comparable with cells cultured in vitro (Fig. 7C), and
IDH2 R172K/A174D or R140Q mutant cells failed to
induce xenograft growth (Fig. 7D).
Discussion
The study of IDH mutations in carcinogenesis has been
hampered by a lack of robust model systems. We previously found that IDH mutation blocked adipocyte
differentiation from 3T3-L1 murine fibroblasts (Lu et al.
2012). This finding has been extended to the hematopoietic system with the observations that mutant IDH and
2HG could impair EPO-induced erythrocyte differentiation in an erythroleukemic cell line (Losman et al. 2013)
and that hematopoietic conditional knock-in IDH1 mutant mice had an expansion in early progenitor/stem cell
population (Sasaki et al. 2012b). Nevertheless, none of the
previous studies reported in vivo tumorigenicity as a result of the presence of mutant IDH. We report here that,
using a nontransformed mesenchymal multipotent cell
line, expression of an IDH2 mutant enzyme producing
high levels of 2HG not only arrested cells from differentiating into adipocytic and chondrocytic lineages, but
also resulted in loss of contact inhibition and tumor
formation in vivo. It is noteworthy that 10T cells have
been frequently used as an in vitro model to test the
carcinogenic potential of chemicals in mesenchymal
cells (for review, see Schechtman 2012). While it is
possible that pre-existing genetic and epigenetic alterations in 10T cells render them more susceptible to 2HGinduced transformation, the in vivo tumorigenicity established here depends on the introduction of a 2HG-producing IDH2 R172K mutant transgene. The morphology
of the mutant IDH2-induced 10T tumors in vivo resembles that of human chondrosarcomas in which the block to
cartilage formation becomes more pronounced as the
tumor progresses. This is consistent with the in vitro
impairment in cell differentiation exhibited by 10T cells
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Figure 6. IDH2 mutant cells show loss of contact inhibition. (A) 10T vector (Vec), wild-type (WT), or R172K mutant IDH2 cells were
cultured in Dulbeccos modified Eagles medium (DMEM), and cell numbers were counted at days 0, 2, 4, and 6. Cells reached
confluence between days 2 and 4. (B) Vector, wild-type, or R172K mutant IDH2 cells at sparse or post-confluence day 2 were incubated
with EdU for 4 h. Percentage of EdU-positive cells was measured by flow cytometry. Histograms from a representative experiment from
a total of two experiments are shown. (C) Vector, wild-type, or R172K mutant IDH2 cells were lysed at sparse, confluence, or 2 d postconfluence. Protein levels of cyclin D1 and p27 were measured by Western blot. Tubulin was used as loading control. (D) 10T cells
expressing wild-type or various mutant IDH2s at post-confluence day 2 were incubated with EdU for 4 h. Percentage of EdU-positive
cells was measured using flow cytometry. Cells were also lysed, and cyclin D1 levels were measured by Western blot. Tubulin was used
as loading control. (E) Vector, wild-type, or R172K mutant IDH2 cells were lysed at sparse or 2 d post-confluence. Levels of N-cadherin
protein expression were measured by Western blot, and mRNA expression was measured by qRT-PCR. For all experiments, the
average 6 SD from three biological replicates are shown.
1993
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Lu et al.
Figure 7. IDH2 mutant cells generate mesenchymal tumors in vivo. (A) We injected
1 3 107 10T vector (Vec), wild-type (WT), or
R172K mutant IDH2 cells subcutaneously
into nude mice. Tumor growth was monitored and measured. The insert image is
shown for mice implanted with wild-type
cells (left) or mutant cells (right) at the time
of sacrifice. (B) Immunohistochemical staining was performed on R172K mutant IDH2
tumors using specific antibodies, and representative images are shown for sections
stained with hematoxylin and eosin (H&E),
Ki67, cyclin D1, and p27. (C) 2HG levels in
R172K mutant IDH2 tumors and parental or
R172K mutant IDH2 10T cells cultured in
vitro were measured by GC-MS. The ratio of
2HG to citrate is shown. (D) We injected 1 3
107 10T R172K, R172K/A174D, or R140Q
mutant IDH2 cells subcutaneously into
nude mice. Tumor growth was monitored
and measured. For all experiments, the average tumor volumes 6 SD of five mice per
group are shown.
1994
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1995
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Lu et al.
1996
Acknowledgments
We thank members of the Thompson laboratory for technical
help and critical reading of the manuscript. We thank the
Memorial Sloan-Kettering Cancer Center (MSKCC) Molecular
Cytology Core Facility for technical help with the immunohistochemical study, the MSKCC Anti-tumor Assessment Core
Facility for technical help with the mouse xenograft study, and
the Epigenomics Core Facility of Weill Cornell Medical College
for help with ERRBS. This work was supported by the Starr
Cancer Consortium, grants from NCI and NIH (to C.B.T.), a
Specialized Center of Research (SCOR) grant from the Leukemia
and Lymphoma Society (to S.W.L.), and a U01 CTDD award from
the National Cancer Institute (to S.W.L.). S.W.L. is the Goeffrey
Beene Chair of Cancer Biology at MSKCC and an Investigator
in the Howard Hughes Medical Institute. C.C. receives a career
development fellowship from the Leukemia and Lymphoma
Society. P.S.W. was supported in part by the University of
Pennsylvania Medical Scientist Training Program. J.H.H. is
funded by the Stephen McDermott Chair in Surgery.
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Supplemental
Material
References
http://genesdev.cshlp.org/content/suppl/2013/09/24/27.18.1986.DC1.html
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